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dc.contributor.authorWang, Qiongqing
dc.date.accessioned2018-07-12T17:32:39Z
dc.date.available2018-07-12T17:32:39Z
dc.date.issued2002
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 64-02, Section: B, page: 5080.;Advisors: Amy Chang.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3080393
dc.identifier.urihttps://hdl.handle.net/20.500.12202/621
dc.description.abstractPMA1 is an essential gene encoding the yeast plasma membrane [H+]ATPase. A pma1-D378N mutant is a substrate of ER quality control and has a dominant-negative effect on cell growth because both newly synthesized mutant and wild-type Pma1 molecules are retained and degraded in the endoplasmic reticulum (ER). Like other substrates for ER-associated degradation (ERAD), Pma1-D378N is stabilized in mutants defective in components of the ubiquitination machinery. A genetic selection was performed for eps (ER-retained pma1 suppressing) mutants in which the growth defect caused by the D378N allele is suppressed. In an eps1 mutant, both mutant and wild-type Pma1 molecules are allowed to travel to the plasma membrane; however, normal retention of resident ER proteins Shr3 and Kar2 is not perturbed. Eps1 is a novel membrane protein belonging to the protein disulfide isomerase (PDI) family, and Eps1 co-localizes with Pma1-D378N in the ER. In the absence of Pma1-D378N, ER export of wild-type Pma1 is not affected by eps1 deletion, but export of the plasma membrane protein Gas1 is delayed. Co-immunoprecipitation experiment reveals direct interaction between Eps1 and Pma1-D378N but not wild-type Pma1. Like other members in the family, Eps1 also has the conserved catalytic site---the CXXC motif and this motif of Eps1 is essential for the ER retention of Pma1-D378N by Eps1.;Besides releasing Pma1-D378N from ER retention and degradation, eps1 cells are sensitive to canavanine and cadmium, and have elevated unfolded protein response, suggesting increased misfolded proteins in these cells. EPS1 genetically interacts with other components of the ER quality control machinery including UBC7 and CDC48. Based on these observations, we propose that Eps1 may act as a novel membrane-bound chaperone, recognizing misfolded proteins in the ER and target them for degradation.;I also demonstrate that Pma1-D378N hetero-oligomerizes with wild-type Pma1 in vivo, thus also making wild-type Pma1 an ERAD substrate. No functional Pma1 at the cell surface results in lethality. In lcb1-100 cells where sphingoid lipid biosynthesis is defective, Pma1 fails to oligomerize. At a semi-permissive temperature of 30°C, lcb1-100 suppresses pma1-D378N because hetero-oligomerization of mutant and wild-type Pma1 does not occur. Wild-type Pma1 is found at the cell surface under these conditions. However, even in the absence of Pma1-D378N in lcb1-100 cells at 30°C, wild-type Pma1 becomes ubiquitinated and is internalized and degraded in the vacuole. At 37°C, in lcb1-100 cells, Pma1 is no longer targeted to the plasma membrane. These data suggest a requirement for sphingoid lipids synthesis at several levels: Pma1 oligomerization, its targeting to the plasma membrane and the maintenance of its cell surface stability.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.subjectMolecular biology.
dc.titleER quality control of the plasma membrane ATPase in Saccharomyces cerevisiae
dc.typeDissertation


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